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Decades of culture-independent analyses have resulted in proposals of many tentative archaeal phyla with no cultivable representative. Members of DPANN (an acronym of the names of the first included phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota), an archaeal superphylum composed of at least 10 of these tentative phyla, are generally considered obligate symbionts dependent on other microorganisms. While many draft/complete genome sequences of DPANN archaea are available and their biological functions have been considerably predicted, only a few examples of their successful laboratory cultivation have been reported, limiting our knowledge of their symbiotic lifestyles. Here, we investigated physiology, morphology, and host specificity of an archaeon of the phylum “Candidatus Micrarchaeota” (ARM-1) belonging to the DPANN superphylum by cultivation. We constructed a stable coculture system composed of ARM-1 and its original host Metallosphaera sp. AS-7 belonging to the order Sulfolobales. Further host-switching experiments confirmed that ARM-1 grew on five different archaeal species from three genera—Metallosphaera, Acidianus, and Saccharolobus—originating from geologically distinct hot, acidic environments. The results suggested the existence of DPANN archaea that can grow by relying on a range of hosts. Genomic analyses showed inferred metabolic capabilities, common/unique genetic contents of ARM-1 among cultivated micrarchaeal representatives, and the possibility of horizontal gene transfer between ARM-1 and members of the order Sulfolobales. Our report sheds light on the symbiotic lifestyles of DPANN archaea and will contribute to the elucidation of their biological/ecological functions.

Tetrathionate hydrolase (TTH) is a key enzyme for the oxidation of reduced inorganic sulfur compounds (RISCs) with the S4I pathway, which is distributed in autotrophic or facultative autotrophic sulfur-oxidizing bacteria and archaea. In this study, the enzyme TTHMc from the acidothermophilic archaeon Metallosphaera cuprina Ar-4T, encoded by mcup_1281 and belonging to the pyrroloquinoline quinone (PQQ) family, has been shown to possess tetrathionate hydrolysis activity. The molecular mass of the single subunit of TTHMc was determined to be 57 kDa. TTHMc is proved to be located in the cytoplasm, periplasmic space, and membrane, and the activity of them accounted for 72.3%, 24.0%, and 3.7% of the total activity. Optimal activity was observed at temperatures above 95 °C and pH 6.0, and the kinetic constants Km and Vmax were 0.35 mmol/L and 86.3 μmol/L, respectively. The presence of 0.01 mol/L Mg2+ enhances the activity of TTHMc, while 0.01 mol/L Ca2+ inhibits its activity. The hydrolysis of tetrathionate (TT) by TTHMc results in the production of thiosulfate, pentathionate, and hexathionate. This study represents the first description of TTH in the genus Metallosphaera, providing new theoretical insights into the study of sulfur-oxidizing proteins in acidothermophilic archaea.

The assimilation of carbon dioxide (CO₂) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO₂ with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO₂-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae , Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum , whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus , Acidianus, and Metallosphaera . Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.

Abstract Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile and of great relevance in bioleaching. However, the impacts of extreme thermoacidophiles propagated with different energy substrates on subsequent bioleaching of refractory chalcopyrite remain unknown. Transcriptional responses underlying their different bioleaching potentials are still elusive. Here, it was first showed that M. sedula inocula propagated with typical energy substrates have different chalcopyrite bioleaching capabilities. Inoculum propagated heterotrophically with yeast extract was deficient in bioleaching; however, inoculum propagated mixotrophically with chalcopyrite, pyrite or sulfur recovered 79%, 78% and 62% copper, respectively, in 12 days. Compared with heterotrophically propagated inoculum, 937, 859 and 683 differentially expressed genes (DEGs) were identified in inoculum cultured with chalcopyrite, pyrite or sulfur, respectively, including upregulation of genes involved in bioleaching-associated metabolism, e.g., Fe2+ and sulfur oxidation, CO2 fixation. Inoculum propagated with pyrite or sulfur, respectively, shared 480 and 411 DEGs with chalcopyrite-cultured inoculum. Discrepancies on repertories of DEGs that involved in Fe2+ and sulfur oxidation in inocula greatly affected subsequent chalcopyrite bioleaching rates. Novel genes (e.g., Msed_1156, Msed_0549) probably involved in sulfur oxidation were first identified. This study highlights that mixotrophically propagated extreme thermoacidophiles especially with chalcopyrite should be inoculated into chalcopyrite heaps at industrial scale.

Nucleoid organization in Crenarchaeota is mediated by a plethora of diverse families of small DNA‐binding proteins. However, the role of the Lrs14 family, a prevalent family of small DNA‐binding proteins within the Crenarchaeal order of the Sulfolobales, remained rather ambiguous. Previous studies have focused on gene regulatory functions of the Lrs14 family and have shown that the Lrs14‐type protein AbfR1 is involved in the regulation of biofilm formation and motility in the model species Sulfolobus acidocaldarius. In this study, we set out to investigate the DNA‐binding characteristics of the AbfR1 homolog in Metallosphaera sedula, a related mixotrophic species within Crenarchaeota. AbfR1Ms and AbfR1Sa share 50% amino acid sequence identity and are structurally very similar. We observed that heterologously purified AbfR1Ms forms dimers in solution and binds DNA in vitro in a non‐sequence‐specific manner with diverse DNA probes. Chromatin immunoprecipitation combined with high‐throughput sequencing revealed an association of AbfR1Ms with numerous sites across the genome of M. sedula. This genome‐wide association was found to correlate with adenine‐thymine‐rich regions and possibly with the global chromatin structure, rather than with specific DNA sequences. Notably, the most highly enriched AbfR1Ms binding sites were characterized by extended DNA regions spanning several thousand base pairs. Atomic force microscopy further demonstrated that AbfR1Ms promotes DNA condensation and aggregation, suggesting a role in chromatin architecture. These findings suggest that AbfR1Ms, and possibly other related Lrs14 members, play a critical role in nucleoid organization, with properties resembling those of bacterial nucleoid‐associated proteins. Nucleoid organization in Crenarchaeota involves diverse small DNA‐binding proteins. The Lrs14‐type protein AbfR1 from Metallosphaera sedula binds nonsequence specifically across the genome and induces DNA condensation. These findings suggest a structural role for AbfR1Ms in chromatin architecture, functionally resembling bacterial nucleoid‐associated proteins.

Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73-0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41-95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.

Metallosphaera sedula is a thermoacidophilic autotrophic archaeon known to utilize the 3-hydroxypropionate/4-hydroxybutyrate cycle (3-HP/4-HB cycle) as carbon fixation pathway. 3-Hydroxypropionyl-CoA dehydratase (3HPCD) is an enzyme involved in the 3-HP/4-HB cycle by converting 3-hydroxypropionyl-CoA to acryloyl-CoA. To elucidate the molecular mechanism of 3HPCD from M. sedula ( Ms 3HPCD), we determined its crystal structure in complex with Coenzyme A (CoA). Ms 3HPCD showed an overall structure and the CoA-binding mode similar to other enoyl-CoA hydratase (ECH) family enzymes. However, compared with the other ECHs, Ms 3HPCD has a tightly formed α3 helix near the active site, and bulky aromatic residues are located at the enoyl-group binding site, resulting in the enzyme having an optimal substrate binding site for accepting short-chain 3-hydroxyacyl-CoA as a substrate. Moreover, based on the phylogenetic tree analysis, we propose that the 3HPCD homologues from the phylum Crenarchaeota have an enoyl-group binding pocket similar to that of bacterial short-chain ECHs.

Metallosphaera sedula is a thermoacidophilic archaeon and has an incomplete TCA/glyoxylate cycle that is used for production of biosynthetic precursors of essential metabolites. Citrate synthase from M. sedula (MsCS) is an enzyme involved in the first step of the incomplete TCA/glyoxylate cycle by converting oxaloacetate and acetyl-CoA into citrate and coenzyme A. To elucidate the inhibition properties of MsCS, we determined its crystal structure at 1.7 Å resolution. Like other Type-I CS, MsCS functions as a dimer and each monomer consists of two distinct domains, a large domain and a small domain. The oxaloacetate binding site locates at the cleft between the two domains, and the active site was more closed upon binding of the oxaloacetate substrate than binding of the citrate product. Interestingly, the inhibition kinetic analysis showed that, unlike other Type-I CSs, MsCS is non-competitively inhibited by NADH. Finally, amino acids and structural comparison of MsCS with other Type-II CSs, which were reported to be non-competitively inhibited by NADH, revealed that MsCS has quite unique NADH binding mode for non-competitive inhibition.